The extracellular matrix or “ECM” is the extracellular part of animal tissue. Due to its diverse nature and composition, the ECM can serve many functions. Functions include providing support, segregating tissues from one another, regulating intercellular communication, and the cell's dynamic behavior. To make matters even more complex, the structure and composition of the ECM are constantly changing in response to the current metabolic activity of the resident cell population, the mechanical demands of the tissue, and the prevailing microenvironmental niche conditions. This concept of “dynamic reciprocity” between the ECM and the resident cell population is a major advantage for the use of ECM scaffold materials over synthetic materials and emphasizes the importance of maintaining as much of both the native composition and its ultrastructure as possible during the preparation of three-dimensional scaffolds.
Components of the ECM are produced intracellularly by resident cells, and secreted (and/or accumulated) into the ECM via exocytosis or cell death. Once secreted they then aggregate with the existing matrix. The ECM is composed of an interlocking mesh of glycosaminoglycans (GAGs) and fibrous proteins, which are discussed below.
GAGs are carbohydrate polymers and are usually attached to extracellular matrix proteins to form proteoglycans (hyaluronic acid is a notable exception, see below). Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+) which attracts water molecules via osmosis, keeping the ECM and resident cells hydrated. Proteoglycans may also help to trap and store growth factors within the ECM.
Different types of proteoglycan found within the extracellular matrix. Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues. It occurs as a proteoglycan (PG) in which two or three HS chains are attached in close proximity to cell surface or extracellular matrix proteins. It is in this form that HS binds to a variety of protein ligands and regulates a wide variety of biological activities, including developmental processes, angiogenesis, blood coagulation and tumour metastasis.
In the extracellular matrix, especially basement membranes, the multi-domain proteins perlecan, agrin and collagen XVIII are the main proteins to which heparan sulfate is attached.
Chondroitin sulfates contribute to the tensile strength of cartilage, tendons, ligaments and walls of the aorta. They have also been known to affect neuroplasticity.
Keratan sulfates have a variable sulfate content and unlike many other GAGs, do not contain uronic acid. They are present in the cornea, cartilage, bones and the horns of animals.
Hyaluronic acid (or “hyaluronan”) is a polysaccharide consisting of alternative residues of D-glucuronic acid and N-acetylglucosamine, and unlike other GAGs it is not found as a proteoglycan. Hyaluronic acid in the extracellular space confers upon tissues the ability to resist compression by providing a counteracting turgor (swelling) force by absorbing significant amounts of water. Hyaluronic acid is thus found in abundance in the ECM of load-bearing joints. It is also a chief component of the interstitial gel. Hyaluronic acid is found on the inner surface of the cell membrane and is translocated out of the cell during biosynthesis. It acts as an environmental cue that regulates cell behavior during embryonic development, healing processes, inflammation and tumor development, and interacts with a specific transmembrane receptor, CD44.
Collagens are the most abundant protein in the ECM in most animals. In fact, collagen is the most abundant protein in the human body and accounts for 90% of bone matrix protein content. Collagens are present in the ECM as fibrillar proteins and give structural support to resident cells. Collagen is exocytosed in precursor form (procollagen), which is then cleaved by procollagen proteases to allow extracellular assembly.
The collagen can be divided into several families according to the types of structure they form:                Fibrillar (Type I, II, III, V, XI)        Facit (Type IX, XII, XIV)        Short chain (Type VIII, X)        Basement membrane (Type IV)        Other (Type VI, VII, XIII)        
Elastins, in contrast to collagens, give elasticity to tissues, allowing them to stretch when needed and then return to their original state. This is useful in blood vessels, the lungs, in skin, and the ligamentum nuchae, and such tissues contain high amounts of elastins. Elastins are synthesized by fibroblasts and smooth muscle cells. They are very insoluble, and tropoelastins are secreted inside a chaperone molecule, which releases the precursor molecule upon contact with a fiber of mature elastin. Tropoelastins are then deaminated to become incorporated into the elastin strand.
Fibronectins are glycoproteins that connect cells with collagen fibers in the ECM, allowing cells to move through the ECM. Fibronectins bind collagen and cell surface integrins, causing a reorganization of the cell's cytoskeleton and facilitating cell movement. Fibronectins are secreted by cells in an unfolded, inactive form. Binding to integrins unfolds fibronectin molecules, allowing them to form dimers so that they can function properly. Fibronectins also help at the site of tissue injury by binding to platelets during blood clotting and facilitating cell movement to the affected area during wound healing.
Laminins are proteins found in the basal laminae of virtually all animals. Rather than forming collagen-like fibers, laminins form networks of web-like structures that resist tensile forces in the basal lamina. They also assist in cell adhesion. Laminins bind other ECM components such as collagens, nidogens, and entactins.
Many cells bind to components of the extracellular matrix. Cell adhesion can occur in two ways; by focal adhesions, connecting the ECM to actin filaments of the cell, and hemidesmosomes, connecting the ECM to intermediate filaments such as keratin. This cell-to-ECM adhesion is regulated by specific cell surface cellular adhesion molecules (CAM) known as integrins. Integrins are cell surface proteins that bind cells to ECM structures, such as fibronectin and laminin, and also to integrin proteins on the surface of other cells.
Fibronectins bind to ECM macromolecules and facilitate their binding to transmembrane integrins. The attachment of fibronectin to the extracellular domain initiates intracellular signaling pathways as well as association with the cellular cytoskeleton via a set of adaptor molecules such as actin.
There are many cell types that contribute to the development of the various types of extracellular matrix found in plethora of tissue types. However, fibroblasts are the most common cell type in connective tissue ECM, in which they synthesize, maintain and provide a structural framework. Fibroblasts also secrete the precursor components of the ECM, including the ground substance. Other cell types include stem cells, as well as chondrocytes and osteoblasts, among others. Chondrocytes are found in cartilage and produce the cartilagenous matrix. Osteoblasts are responsible for bone formation.
The ECM is of intense medical and scientific interest because it can be used to provide a scaffold for tissue growth and regeneration. Because native ECM guides organ development, repair and physiologic regeneration, it provides a promising alternative to synthetic scaffolds and a foundation for regenerative efforts.
In the last few years, studies have confirmed the importance of ECM in regulating stem cells differentiation in mature tissue. Many of the factors responsible for the influence of ECM on stem cells are related to its properties, including matrix structure, composition, elasticity, and integrity. Because these ECM properties are often tissue type specific, in order for developing differentiated cells from stem cells for therapeutic applications, the capability of generating specific ECM types may be of great value to regulate stem cell environments (Reilly, 2010).
One of the recent paradigm shifts in stem cell biology has been the discovery that stem cells can begin to differentiate into mature tissue cells when exposed to intrinsic properties of the extracellular matrix (ECM), such as matrix structure, elasticity, and composition. These parameters are known to modulate the forces a cell can exert upon its matrix. Mechano-sensitive pathways subsequently convert these biophysical cues into biochemical signals that commit the cell to a specific lineage. Just as with well-studied growth factors, ECM parameters are extremely dynamic and are spatially- and temporally-controlled during development, suggesting that they play a morphogenetic role in guiding differentiation and arrangement of cells. Our ability to dynamically regulate the stem cell niche the way it naturally occurs in the body is likely to be a critical requirement for developing differentiated cells from stem cells for therapeutic applications.
ECM has been prepared by harvesting an appropriate tissue and decellularizing it with chemical and/or enzymatic means of cell lysis, and/or physical means of stripping cells, such as forced flow over or through the tissue. However, these methods require a lot of time, provide low yields, and usually a patient cannot provide such tissue, necessitating resource to cadavers or animal tissues. Thus, the existing methodology is not ideal.
Another method of preparing ECM is to make it from cells grown in culture. Cells gown in monolayer have been used to make ECM, but the technique is difficult and subject to very low yields. Further, 2D cultures do not fully and accurately reflect the organization and structure of native ECM, again making such methods less than ideal.
Although some of these approaches are promising, there is still room for improved methods of preparing and using ECM that avoids some of the disadvantages of the prior art and provides high yield ECM that is as close to native ECM as possible.